Caveolin-1 Genetic Polymorphisms Interact with Fatty Acid Types to Modulate Metabolic Syndrome Risk

Background: Metabolic syndrome (MetS) is related with all-cause mortality. Caveolin-1 (Cav-1) has been widely studied in dyslipidemia, and several studies have indicated that Cav-1 genetic variations may correlate with dietary intake of fatty acids. The aim of the current study was therefore to evaluate the interaction of Cav-1 rs3807992 with types of dietary fatty acid in MetS risk factor status Methods: This cross-sectional study was carried out on 404 overweight and obese females. Dietary intake was obtained from a 147-item FFQ. The CAV-1 genotype was measured using the PCR-RFLP method. Anthropometric values and serum levels (TC, LDL, HDL, TG, FBS) were measured by standard methods. Results: It was observed that the (AA+AG) group had signicantly higher BMI, WC and DBP (P=0.02, P=0.02 and P=0.01, respectively) and lower serum LDL, HDL and TC (P < 0.05) than the GG group. It was found that A allele carriers were at higher odds of MetS (P= 0.01), abdominal obesity (P=0.06), increased TG concentration (P=0.01), elevated blood pressure (BP) (P=0.01), increased glucose concentration (P=0.45), and decreased HDL-cholesterol concentration (P=0.03). Moreover, the interaction of Cav-1 and SFA intake was signicant in terms of MetS (P=0.01), LDL (P=0.03), DBP (P=0.01) and LDL/HDL (P=0.05). Additionally, the (AA+AG) group was signicantly related to PUFA intake in terms of MetS (P=0.04), TG (P=0.02), glucose (P=0.02) and HOMA-IR (P= 0.01). Conclusions: Higher PUFA consumption might attenuate the Cav-1 rs3807992 associations with MetS, and individuals genetic have a higher risk of MetS, associated with higher SFA consumption To date, studies on this polymorphism have been animal studies and have not been performed on healthy and obese human society For the rst time , this study provides information on the interaction of different fatty acids with the Caveolin gene, which is functionally effective in lipid metabolism polymorphisms genotype


Introduction
Metabolic syndrome (MetS) is related with metabolic abnormalities including obesity, hyperglycemia, dyslipidemia, and hypertension, and is prevalent worldwide 1 . MetS is related to all-cause mortality, myocardial infarction (MI), and stroke in patients with or without diabetes 2 . Surprisingly, the evidence demonstrates that genetic variations play a main part in the prevention and treatment of various chronic disease, particularly in MetS [3][4][5] . MetS and cardiovascular diseases (CVD) also demonstrate the role of genetic and environmental factors in diet-related disorders. A genetic background of metabolic disorders has been shown to relate to death rate over the last few decades 6 . There is therefore a need to identify the genes that derive MetS and to develop new therapies. Caveolin-1 (Cav-1) is a key protein component of caveolae, and has been widely studied in dyslipidemia and CVD due to signal transduction, tra cking in cholesterol hemostasis, and triacylglycerol metabolism 7 . Mice treated with Cav-1 are resistant to highfat diets, and they show lipodystrophy, hypertension, insulin resistance, and abnormal glucose metabolism 8 . Moreover, several studies have indicated that Cav-1 genetic variations might interact with other risk factors, including dietary intake of fatty acids, suggesting a positive association between Cav-1 and hypercholesterolemia 9 . In studies conducted among Caucasian and Hispanic cohorts, the prevalence of Cav-1 gene variant rs926198 is related with higher odds of MetS risk and low HDL 10,11 .
Also, Cav-1 overexpression has been observed to relate to higher odds of atherosclerosis in experimental models 12 . The exact mechanisms are unclear, but it seems that Cav-1 is able to regulate several key enzymes in lipid metabolism, such as cholesterol ester transfer protein and phospholipid transfer protein 13 . While the association between Cav-1 polymorphisms and type 2 diabetes risk has been widely reported in various populations 14 , these relationships with MetS have been inconsistent, despite several publications on the association between Cav-1 gene variants and serum lipid pro les [14][15][16][17] . To the authors' knowledge, there has been no study evaluating Cav-1 rs3807992 variant, metabolic risk factor, and the interaction of fatty acid intake levels with this SNP. Hence, the aim of the current study was to evaluate the interaction of Cav-1 genetic polymorphism with the types of dietary fatty acids, in terms of MetS risk factor status.

Study Population
A total of 404 women in the range of 18-55 years old were randomly selected from among participants in a cross-sectional study performed in 2019.Participants provided written informed consent. The inclusion criteria were: obese or overweight, no alcohol consumption, and no smoking. The exclusion criteria were: CVDs, kidney failure, stroke, thyroid disease, liver disease, cancer, in ammatory illnesses, and those taking any therapeutic medications. Each participant was interviewed in order to obtain demographic data, then referred to the laboratory for blood sampling. Anthropometric measures were taken, including: height (m), weight (kg), waist circumference (WC, cm) measured at the narrowest part of the abdomen, and body mass index (BMI, kg/m 2 , calculated by dividing weight by height squared). Blood pressure (BP) was measured with a sphygmomanometer (BP) after 5 minutes' rest. Triglyceride (TG) (mg/dl), highdensity lipoprotein (HDL) (mg/dl), total cholesterol (mg/dl), and low-density lipoprotein (LDL) (mg/dl) were measured according to standard protocols. The study was approved by the Ethics Committee at the Tehran University of Medical Sciences (TUMS) (97-03-161-41017).

MetS De nition
MetS cases were required to meet three or more of the following criteria according to the Adult Treatment Panel III (ATP III) criteria: 1) Elevated fasting blood glucose FPG≥100 mg/dl.

Genotyping
For genotyping the Cav-1 polymorphisms, DNA was extracted from whole blood via a Mini Columns kit (Type G; Genall; Exgene). The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was employed to investigate Cav-1 polymorphisms (rs3807992) in gene fragments (major allele G and minor allele A). PCR was carried out using the following primers: F:3′AGTATTGACCTGATTTGCCATG5′ R:5′GTCTTCTGGAAAAAGCACATGA-3′. PCR reactions were performed in a volume of 20 µl, containing 1 µl extracted DNA, 1 µl Forward primers, 1 µl Reverse primers ,7 µl distilled water and 10 µl Taq DNA Polymerase Master Mix in a DNA thermocycler. The DNA templates were denatured at 94 °C for 3 min and 40 cycles, including a min denaturation at 94 °C, a min annealing at 42-50 °C and elongation at 72 °C for 2 min. Ampli ed DNA was digested with Hin1II (NIaIII) restriction enzyme at 37 °C overnight, then separated by electrophoresis on an agarose gel (2%).

Statistical Analysis
Statistical analysis was performed using SPSS v.25 software (SPSS Inc., IL, USA) and signi cance level was considered P < 0.05. The Kolmogorov-Smirnov was utilized to test the normality of the data, and all data were expressed as means ± SD. The Pearson's chi-square test was used to determine the Hardy-Weinberg Equilibrium deviation among allele frequency of Cav-1 G32124A (rs3807992). Independentsample t-test was used to evaluate the differences between the two groups, and ANCOVA test was used to evaluate for confounding effects. Binary logistic regression was used to estimate interactions between rs3807992 and high and low dietary fat intake on the Odds Ratio (OR) of the MetS component. All ORs were adjusted for variables proven to be related to MetS components, (such as age, educational level, smoking status, physical activity and energy intake).

Dietary Assessment
The Food Frequency Questionnaire (FFQ) was a useful tool for evaluating dietary intake, and included 147 items 19 . This assessment was carried out by interviewing the occurrence of food items consumed on the basis of a predetermined list of foods. The extracted FFQ values were then changed to grams/day.
For the evaluation of macro-and micronutrient content, N4 software was used; all measurements were then entered into IBM SPPS.

Results
Clinical Characteristics According to Cav-1 rs3807992 Genotypes As shown in Table 1, 404 women with a mean age of 36.67 years were evaluated in the present study. The results of dietary nutritional intake are described according genotype groups, which show that the (AA+AG) group had increased SFA and cholesterol consumption (g/d), compared to the GG group. No signi cant differences were found in other nutrient consumption values ( age at onset of obesity, and total PUFA intake). There was no further signi cant interaction between the (AA+AG) group and SFA intake on other biochemical parameters, including HDL, TG, TC and LDL/HDL, in both the crude and adjusted models (P >0.05) (

Discussion
According to these ndings, female A-allele carriers had signi cantly higher BMI, WC and DBP, and had lower serum LDL, HDL and TC, compared to GG genotypes. This demonstrates that their clinical parameters are predisposed to MetS. A-allele carriers were at higher odds of MetS. To the authors' knowledge, there is little evidence describing an association between Cav-1 and metabolic syndrome. It was observed that a genetic variant of Cav-1 (rs3807992) was associated with increased MetS risk, which is consistent with previous studies that revealed a signi cant association between the minor allele in cav-1 variations and the odds of metabolic diseases 10,11,15,17 . Also, compared with other candidate gene studies, no studies had evaluated SNP rs3807992 and MetS risk. It is proposed that Cav-1 polymorphisms increase MetS risk through altered Cav-1 gene expression, attenuating dyslipidemia and hypertension, while impairing glucose and insulin homeostasis [12][13][14]20 . Cav-1 regulates signaling molecules, such as IRS1, that have a key role in appropriate insulin responses, PKA, angiotensin II receptors, active blood pressure molecules, and binding sites for calcium ions; all of these may affect various clinical traits of MetS 14,21,22 . Additionally, Cav-1 is able to effect NO, insulin, lipids, and hormone metabolisms 23 . Therefore, caveolae and its components may become useful sites for further investigation into treating MetS.
The genetic association between Cav-1 polymorphisms and MetS by dietary fat intake was reported. High dietary SFA intake (≥25gr) especially accentuated the negative effects of rs3807992 in terms of MetS risk. Furthermore, a potential gene-nutrient interaction was found between the rs3807992 Cav-1 polymorphism and PUFA intake. High PUFA intake (≥6% energy) reduced the negative effects of rs3807992 in terms of MetS risk, with the greatest protection achieved by A-allele carriers. To the authors' knowledge, this interaction has not been previously studied, and the present paper's nding support the necessity of lower and higher consumption of SFA and PUFA, respectively, in the diets of A-allele carriers.
Most previous intervention studies have shown that a higher intake of SFA was detrimental to maintaining insulin sensitivity, whereas PUFA showed bene cial effects 24 . Additionally, environmental factors, in particular dietary composition, may alter the risk of MetS. Nutrigenetic research has indicated that dietary fat background can in uence genotype-phenotype relations 25 .
In line with previous studies, experimental studies have reported that sphingomyelin is a key phospholipid of caveolae. SFA intake may lead to increases in sphingolipids levels in the cardiac cell membranes, thus disrupting the caveolae contents 26,27 . Moreover, Chapkin et al. reported that in animal models, n-3 PUFA intake may modulate the function of caveolae proteins/lipid, affecting membrane fusion and cell-cell signaling, and improving insulin signaling. Many studies have indicated that H-Ras and eNOS (endothelial nitric oxide synthase) are moved from caveolae in n-3 PUFA supplemented rats, which suppressed the Ras-dependent signaling and reduced blood pressure, thereby lowering the MetS risk 28 . Caveolae membrane fatty acids (in the internal and external lea et) is also signi cantly altered by n-3 PUFA intake, and is even able to change the function of the caveolae.
Caveolae plays a unique function in the uptake of various lipid and glucose metabolites. Accordingly, caveolae is a main center for several nutrient metabolisms through the cell membrane 29 . Caveolae is able to uptake fatty acid, triacylglycerol, and cholesterol in many tissues, which leads to an elevation in caveolae density in obese rats 30,31 . However, in these animals, the number of caveolae decreases in the arterial endothelium and at the ends of smooth muscle cells, leading to dyslipidemia 32 .
The mechanism observed in the present study, by which fatty acids are able to modify the genetic risk posed by Cav-1 polymorphisms, remains unknown; further studies are needed to indicate such gene-diet interventions. Several limitations can also be identi ed in the current study. Dietary intake was assessed by a food-frequency questionnaire (FFQ), which is self-reported and thus dependent on patient memory. Due to nancial limitations, it was not possible to perform western blot analysis to determine whether rs-3807992 SNP alters the expression of Cav-1. The focus of the current study was on dietary fat composition, but other nutrient components, including carbohydrates or ber, can also play a role in the progression of MetS. Finally, given the observational nature of the study, it is not possible to tell whether the associations which were identi ed in women (but not men) are of a causal nature.

Conclusion
To the authors' knowledge, this is the rst study presenting the association of a genetic variant of Cav-1 rs3807992 with the risk of MetS and its components, including TG, BP, and HDL level. However, further studies are needed to determine the strength of this association in a larger population; the contribution of this study is the novel nding that rs3807992 clearly predicts MetS among obese women. Analyses of the individual components of MetS con rmed that the rs3807992 variant is related to elevated BP, dyslipidemia, low HDL cholesterol, and high TG levels. Also, Cav-1 rs3807992 genotypes are sensitive to dietary SFA and PUFA, which allows individuals to monitor and adjust SFA and PUFA consumption accordingly. Finally, these results can be used in combination with a patient's genetic history in order to provide more applicable and tailored nutritional advice for preventing or attenuating MetS in overweight and obese women.   Comparisons between groups were determined based on logistic regression analysis